The fluorescent dyes PpIX, fluorescein, and ICG are used in the medical field, in particular for staining biological material, for example blood cells, tumors or other tissue. If the tissue stained with a fluorescent dye is illuminated by light with a wavelength lying in the absorption spectrum of the fluorescent dye, the fluorescent dye emits fluorescence light. This fluorescence light may be detected in turn, as a result of which the region of a specific tissue stained with the fluorescent dye may be made visible and may be made distinguishable from the surrounding biological material. Frequently, a plurality of different fluorescent dyes are used for staining different biological materials within the scope of an operation. First of all, the surgeon may consider it advantageous to simultaneously observe different biological materials which were stained with different fluorescent dyes in each case. Secondly, it may likewise be advantageous to make certain biological materials visible by fluorescence light in a selective manner so as not to be distracted by a different fluorescence at the same time.
In the field of surgical applications, fluorescence observation systems are usually integrated in conventional surgical microscopes or provided by the latter. Therefore, as a rule, the fluorescence observation system is arranged over the region to be operated on and it should therefore be as compact as possible, i.e., take up little installation space, so as not to impede the surgeon. By way of example, conventional fluorescence observation systems are embodied as stereo microscopes, where the surgeon can view the operation region through eyepieces. A fluorescence image presented on a display may be overlaid onto the observation beam path of the stereo microscope with the aid of a further optical system, and so the surgeon is able to see the operation region and the fluorescence image at the same time.
In conventional fluorescence observation systems, use is frequently made of a single broadband light source which provides both the excitation light, i.e., the light for exciting the fluorescent dyes, and the illumination light, i.e., the light for producing an overview image. Usually, an illumination filter arranged in the beam path from the light source to an object region, in which the object that was stained with the fluorescent dyes is arranged, is required and it forms the intensity spectrum of the excitation light directed onto the object region and the intensity spectrum of the illumination light directed onto the object region. The illumination filter adopts the object of suitably adjusting the intensity ratios between the excitation light and the illumination light.
As a rule, it is not possible to simultaneously observe a plurality of fluorescences using conventional fluorescence observation systems since the employed filter system, consisting of the illumination filter and a detection filter that is arranged in the beam path from the object region to the eyepieces, is only optimized for a single fluorescent dye.
As shown schematically in FIG. 2A, the fluorescent dye protoporphyrin IX (PpIX) has an absorption spectrum which, between 350 nm and 430 nm, has a normalized absorption intensity of more than 0.2. The normalized absorption intensity is normalized to the maximum absorption intensity; i.e., the normalized absorption spectrum only has values of between 0 and 1. Therefore, the fluorescent dye PpIX may be efficiently excited in the range from 350 nm to 430 nm. The fluorescent dye has the maximum of the absorption at approximately 405 nm. The fluorescent dye PpIX emits fluorescence light in a spectral range from approximately 600 nm to 750 nm, with a main maximum of the emission intensity lying at 635 nm and a secondary maximum lying at approximately 705 nm.
As shown schematically in FIG. 3A, the fluorescent dye fluorescein has, between approximately 450 nm and 530 nm, a normalized absorption intensity of more than 0.2. Therefore, the fluorescent dye fluorescein can be excited well in this range. The absorption spectrum of fluorescein has a maximum at approximately 495 nm. The fluorescent dye fluorescein emits emission light in the range from approximately 490 nm to 650 nm. The maximum of the emission spectrum lies at approximately 520 nm.
As shown schematically in FIG. 5A, the fluorescent dye indocyanine green (ICG) has, between 700 nm and approximately 840 nm, a normalized absorption intensity of more than 0.2. Therefore, indocyanine green can be excited well in this range. The maximum of the absorption spectrum of indocyanine green lies at approximately 800 nm. The fluorescent dye indocyanine green emits emission light in the range from approximately 750 nm to 1000 nm. The maximum of the emission spectrum lies at approximately 835 nm.